Abstract

Opioids mediate their analgesic effects by activating μ-opioid receptors
(MOR) not only within the central nervous system but also on peripheral
sensory neurons. The peripheral analgesic effects of opioids are best
described under inflammatory conditions (e.g., arthritis). The present study
investigated the effects of inflammation on MOR binding and G-protein coupling
of full versus partial MOR agonists in dorsal root ganglia (DRG) of primary
afferent neurons. Our results show that Freund's complete adjuvant (FCA)
unilateral hindpaw inflammation induces a significant up-regulation of MOR
binding sites (25 to 47 fmol/mg of protein) on DRG membranes without affecting
the affinity of either full or partial MOR agonists. In our
immunohistochemical studies, the number of MOR-immunoreactive neurons
consistently increased. This increase was mostly caused by small-diameter
nociceptive DRG neurons. The full agonist DAMGO induced MOR G-protein coupling
in DRG of animals without FCA inflammation (EC50 = 56 nM; relative
Emax = 100%). FCA inflammation resulted in significant
increases in DAMGO-induced MOR G-protein coupling (EC50 = 29 nM;
relative Emax = 145%). The partial agonist buprenorphine
hydrochloride (BUP) showed no detectable G-protein coupling in DRG of animals
without FCA inflammation; however, partial agonist activity of BUP-induced MOR
G-protein coupling was detectable in animals with FCA inflammation
(EC50 = 1.6 nM; relative Emax = 82%). In
behavioral studies, administration of BUP produced significant antinociception
only in inflamed but not in noninflamed paws. These findings show that
inflammation causes changes in MOR binding and G-protein coupling in primary
afferent neurons. They further underscore the important differences in
clinical studies testing peripherally active opioids in inflammatory painful
conditions.

Opioid analgesia is not mediated exclusively within the central nervous
system but also in the periphery. This has been shown in many animal models,
including unilateral hindpaw inflammation induced by intraplantar injection of
Freund's complete adjuvant (FCA) (Stein et
al., 1988a). Moreover, controlled clinical trials have reported
peripheral analgesic effects of opioids in both short-term postoperative and
long-term arthritic pain (Stein et al.,
2001). The peripheral analgesic effects of opioids are elicited by
activation of opioid receptors on primary afferent neurons. This is best
described under local inflammatory conditions
(Stein et al., 1989). In
addition, it has been shown in clinical studies that the effects of exogenous
opioids in peripheral antinociception were enhanced in inflamed tissue
(Stein et al., 2001). It was
suggested that an increase in antinociception during inflammation might be
related to an increase in the number of μ-opioid receptors (MOR) in dorsal
root ganglia (DRG) (Ji et al.,
1995). However, it remains unclear whether inflammation alters
intracellular signaling (e.g., G-protein coupling and ligand binding of MOR on
peripheral sensory neurons). Therefore, this study compares animals with and
without inflammation to examine whether FCA inflammation 1) alters MOR binding
in DRG neurons, 2) leads to immunohistochemical differences in the
distribution and density of MOR on DRG neurons, 3) causes differences in the
potency and efficacy of agonists coupling to MOR of DRG neurons, or 4) reveals
differences in behavioral studies for the partial MOR agonist BUP.

Subjects. Experiments were performed in male Wistar rats
(180–200 g) individually housed in cages lined with sawdust. Standard
laboratory rodent chow and water were available ad libitum. Room temperature
and relative humidity were maintained at 22 ± 0.5°C and 60%,
respectively. A 12-h:12-h light/dark cycle was used. All testing was conducted
in the light phase, employing separate groups of animals. The guidelines on
ethical standards for investigations of experimental pain in animals were
followed (Zimmermann,
1983).

Induction of Inflammation. Unilateral hindpaw inflammation was
induced by injection of 0.15 ml of FCA into the right hindpaw under brief
halothane anesthesia. A detailed description of the time course and magnitude
of the inflammatory reaction is given elsewhere
(Stein et al., 1988b). The
inflammation remained confined to the inoculated paw and all experiments were
performed 96 h (4 days) after FCA inoculation.

Membrane Preparations. Rats were killed by halothane anesthesia
after 96-h treatment with saline or FCA and lumbar (L3–L5) DRGs were
removed. In animals treated with FCA, DRG on the inflamed and contralateral
sites were removed separately. The tissue was placed immediately on ice in
cold assay buffer (50 mM Tris-HCl, 1 mM EGTA, 5 mM MgCl2, pH 7.4).
Membrane preparations were made by pooling DRG tissue from 10 rats. Tissue was
homogenized with a Polytron homogenizer (Kinematica AG, Littau, Switzerland)
and centrifuged at 48,000g at 4°C for 20 min. The pellet was resuspended
in assay buffer followed by a 10-min incubation at 37°C to remove
endogenous ligands. The homogenate was centrifuged again at 48,000 g and
resuspended in assay buffer. Membranes were aliquoted and stored at
-80°C.

Preparation of Sciatic Nerves. Rats were anesthetized with halothane
48 h after FCA or saline injections. The right sciatic nerve was surgically
exposed, dissected away from the surrounding tissue, and ligated with
nonabsorbable silk at the midfemoral position (5 mm below the sciatic notch)
in animals with FCA inflammation or saline treatment. The incision was then
closed with wound clips. After 96 h of FCA inflammation, rats were killed, and
the proximal part of sciatic nerve was removed and membranes prepared as
described above.

Opioid Receptor Binding. Membranes were diluted in assay buffer.
Saturation analysis of [3H] DAMGO binding was performed by
incubating 50 μg of membrane protein with 0.02 to 2 nM [3H]DAMGO
in the presence and absence of 10 μM unlabeled naloxone (NLX) to determine
nonspecific binding. Affinity (inhibition constants, Ki)
of DAMGO and BUP at DRG membranes of animals with and without FCA inflammation
were determined in [3H]DAMGO competition binding experiments. In
animals with FCA inflammation, the contralateral side of DRG was removed and
MOR binding sites were detected by incubating 50 μg of membrane protein
with 2 nM [3H]DAMGO in the presence and absence of 10 μM
unlabeled NLX. The accumulation of MOR binding sites in the sciatic nerve was
detected by incubating 75 μg of membrane protein with 2 nM
[3H]DAMGO in the presence and absence of 10 μM unlabeled NLX.
Membranes were incubated for1hat 30°C in assay buffer. The reactions were
terminated by rapid filtration under vacuum through Whatman GF/B glass fiber
filters, followed by four washes with cold buffer (50 mM Tris-HCl, pH 7.4).
Bound radioactivity was determined by liquid scintillation spectrophotometry
after overnight extraction of the filters in 3 ml of scintillation fluid.

Immunohistochemistry. Four days after FCA treatment, six rats were
deeply anesthetized with halothane and transcardially perfused with 60 ml of
warm saline, followed by 300 ml of 4% (w/v) paraformaldehyde with 0.2% (v/v)
picric acid in 0.16 M phosphate buffer solution, pH 6.9. The ipsilateral and
contralateral L5 DRG were removed, postfixed in the same fixatives for 90 min,
and then placed in 15% (w/v) sucrose solution at 4°C overnight. The tissue
was embedded in Tissue Tek compound (OCT; Miles), frozen and cut in 14-μm
sections. The sections were incubated overnight with anti-MOR (1:1000) (kindly
provided by Drs. Stefan Schulz and Volker Höllt, Department of
Pharmacology and Toxicology, Otto-von-Guericke University, Magdeburg,
Germany). The sections were incubated for 90 min with the appropriate
biotinylated secondary antibody and with avidin-biotin-conjugated peroxidase.
Finally, the sections were washed and stained with
3′,3′-diaminobenzidine tetrahydrochloride containing 0.01%
H2O2 in 0.05 M Tris-buffered saline, pH 7.6, for 3 to 5
min. After the enzyme reaction, the sections were washed in tap water, mounted
onto gelatin-coated slides, dehydrated in alcohol, cleared in xylene, and
mounted in dibutylpthalate polystyrene xylene. To demonstrate specificity of
staining, the following controls were included: 1) preabsorption of diluted
antibody against MOR with a synthetic peptide for MOR (Gramsch Laboratories,
Schwabhausen, Germany) for 24 h at 4°C and 2) omission of either the
primary antisera, the secondary antibodies, or the avidin-biotin complex.
These control experiments did not show MOR staining.

The method of quantification for DRG staining has been described previously
(Ji et al., 1995). Briefly, we
stained every fourth section of DRG that was serially cut at 14 μm. The
total number of MOR-containing neurons was counted by an observer blinded to
the experimental protocol. This number was divided by the total number of
neurons in each DRG section, and the percentage of MOR immunoreactive neurons
was calculated. Percentages from four sections of each DRG were averaged. Five
rats per group (inflamed and noninflamed) were used for analysis. The cell
body diameter was measured with the nucleus in the focal plane and was
estimated from the average length and width determined with a calibrated
micrometer. A total number of 30 immunoreactive neurons with nucleus were
measured in each animal.

Measurement of Agonist Efficacy and Potency at MOR in DRG Membranes.
Membranes were thawed, homogenized, and centrifuged at 48,000g for 10
min. Membranes were resuspended in [35S]GTPγS assay buffer
(50 mM Tris-HCl, pH7.4, 5 mM MgCl2, 0.2 mM EGTA, 100 mM NaCl, and 1
mM DTT). The buffer composition was similar to that used by Newman-Tancredi et
al. (2000).
Concentration-effect curves were generated by incubating the appropriate
concentration of membranes (50 μg) in assay buffer with 0.1% bovine serum
albumin, various concentrations of BUP or DAMGO
(10-12–10-4 M), with 50 μM GDP and 0.05 nM
[35S]GTPγS in a total volume of 800 μl. Basal binding was
assessed in the absence of agonist, and nonspecific binding was measured in
the presence of 10 μM unlabeled GTPγS. The reaction was incubated for
2 h at 30°C.

[35S]GTPγS Saturation Binding at MOR of DRG
Membranes. Saturation analysis of DAMGO and BUP-stimulated
[35S]GTPγS binding to DRG membranes was performed. In the
presence (DAMGO or BUP 10 μM) or absence (H2O) of agonists,
membranes were incubated with 0.05 to 2 nM [35S]GTPγS in
assay buffer for 2 h at 30°C. Unstimulated [35S]GTPγS
binding was subtracted from agonist-stimulated binding at each measurement
point. The incubations for all experiments were terminated by filtration under
vacuum through Whatman GF/B glass fiber filters, followed by four washes with
cold buffer (50 mM Tris-HCl, pH 7.4). Bound radioactivity was determined by
liquid scintillation spectrophotometry after extraction overnight in
scintillation fluid.

Measurement of Paw Pressure Threshold. Four days after FCA
inoculation, nociceptive thresholds were assessed before (baseline) and after
drug administration using the paw pressure algesiometer (modified
Randall-Selitto test; Ugo Basile, Comerio, Italy). The pressure required to
elicit paw withdrawal, the paw pressure threshold (PPT) (cutoff at 250 g), was
determined by averaging three consecutive trials separated by 10 s
(Stein et al., 1988b). The
sequence of left and right paws was alternated between animals to avoid bias.
Drugs were administered intraplantarly (100 μl), and antagonists were given
concomitantly with agonist in a total volume of 200 μl. Control animals
received saline in the same volume. The experimenter was blind to the
treatment.

Data Analysis. All ligand binding and [35S]GTPγS
binding data are reported as mean ± S.E. values of at least three
experiments, each of which was performed in duplicate. [3H]DAMGO
ligand binding experiments and [35S]GTPγS saturation binding
experiments were fitted to a one-site binding hyperbola using Prism (GraphPad,
San Diego) to determine Kd and Bmax
values. For saturation analysis of stimulated [35S]GTPγS
binding, basal [35S]GTPγS binding was subtracted from agonist
(10 μM DAMGO or 10 μM BUP)-stimulated [35S]GTPγS
binding. Stimulated [35S]GTPγS binding is defined as
agonist-stimulated minus basal [35S]GTPγS binding. Efficacy
(Emax) is defined as the maximum percentage stimulation by
an agonist, as determined by nonlinear regression analysis of
concentration-effect curves. Relative Emax values are
expressed as a percentage of maximal stimulation with DAMGO in animals without
inflammation. Nonspecific binding was subtracted from all
[35S]GTPγS binding data. Statistical differences between
animals with and without FCA inflammation were determined by the nonpaired
Student's t test and Mann-Whitney rank sum tests. Amplification
factors were defined by DAMGO-activated G-protein Bmax/MOR
Bmax. Behavioral data are represented as mean ±
S.E.M. Dose-response curves were assessed by analysis of variance followed by
a post hoc Dunnett test. Time course data were analyzed by two-way
repeated-measure analysis of variance (treatment × time) followed by a
post hoc Dunnett test. Differences were considered significant at p
< 0.05. All tests were performed using Sigma Stat 2.03 (SPSS Science,
Chicago, IL) statistical software

A, saturation binding was performed with [3H]DAMGO to DRG
membranes of animals with and without inflammation. Nonspecific binding was
determined with 10 μM naloxone. Data shown are means of duplicates from at
least three independent experiments. B, displacement of [3H]DAMGO
binding to DRG membranes of animals with and without inflammation.
Nonradioactive DAMGO and BUP was tested as a displacer at 10-11 to
10-4 M concentrations. One representative curve of four independent
experiments is shown.

MOR in Sciatic Nerve Membrane Preparations. In the sciatic nerve of
unligated rats, almost no specific binding was detectable (data not shown). In
the absence of inflammation, an accumulation of binding sites was shown
proximal to the ligature (7 ± 1.3 fmol/mg of protein). However, after
96 h of FCA inflammation, a significantly higher accumulation of MOR-specific
binding sites was detected in proximal parts of the ligature (17 ± 2.5
fmol/mg of protein, t test, p < 0.05).

Immunohistochemistry. In nontreated rats, some DRG neurons contained
MOR-immunoreactivity (MOR-IR) (Fig.
2A). These neurons were mainly of small diameter. The mean cell
body diameter of MOR-positive neurons was 37.4 + 1.1 μm; the majority lay
between small- and medium-diameter neurons (23–51 μm). Of all
neurons, 17.2 ± 0.9% were MORIR. Four days after FCA, there was a
noticeable increase in the number of MOR-positive DRG neurons on the inflamed
side (Fig. 2B) and this
increase in MOR was not detectable on the contralateral side of inflammation
(data not shown). Of all DRG neurons, 25 ± 1.3% were MOR-positive,
which represents a 45.3% relative increase (p < 0.01, Mann-Whitney
rank sum test). There was no significant difference in the mean diameter of
MOR-positive neurons between animals with and without FCA inflammation
(p > 0.05), suggesting that any increase in MOR synthesis was not
caused by a change in cell size.

Bright-field micrographs showing MOR positive neurons in L5 DRGs of rats
without FCA inflammation (A) and in DRGs of rats with FCA inflammation (B).
MOR-IR is mainly seen in small DRG neurons. Scale bar, 20 μm.

Potencies and Efficacies of DAMGO and BUP for
[35S]GTPγS Binding in DRG. It has been shown that
agonist efficacy for stimulation of [35S]GTPγS binding is
dependent on the concentration of GDP
(Traynor and Nahorski, 1995;
Selley et al., 1997;
Newman-Tancredi et al., 1999).
Among various concentrations (5–200 μM) of GDP tested, 50 μM GDP
achieved the maximal percentage stimulation by DAMGO in DRG membranes and was
used in all subsequent studies. EC50 and Emax
values are shown in Table 1 and
Fig. 3. Relative
Emax values were expressed as a percentage of maximal
DAMGO stimulation in DRG membranes of animals without FCA inflammation. After
96 h of FCA inflammation, DAMGO induced a significant increase in efficacy
(Emax) (Mann-Whitney rank sum test, p < 0.05)
and a nonsignificant, leftward shift in potency in DRG membranes
(Table 1,
Fig. 3A). In contrast, the
partial agonist BUP did not induce any detectable G protein activation in DRG
membranes of animals without FCA inflammation. However, after 96 h of FCA
inflammation, BUP showed effective G-protein coupling
(Table 1,
Fig. 3B).

[35S]GTPγS intrinsic efficacies
(Emax), potencies (EC50), and relative
Emax of DAMGO and BUP in DRG membranes of animals without
(Control) and with (FCA) inflammation

Data are mean values ± S.E.M. of at least three independent
experiments as described in Materials and Methods. Emax
values are percentage stimulation over basal and relative
Emax values are expressed as a percent of maximal
stimulation with DAMGO in animals without inflammation.

Stimulation of [35S]GTPγS binding to DRG membranes of
animals without (Control) and with (FCA) inflammation. Concentration-response
curves were determined for DAMGO (A) and BUP (B), as described under
Materials and Methods. [35S]GTPγS binding is
expressed as percentage of maximal stimulation with DAMGO (100%) obtained in
noninflamed animals. Nonspecific binding was determined using 10 μM
unlabeled GTPγS and was subtracted from each data set. Basal
[35S]GTPγS binding in the absence of added drugs was 4000 to
6000 cpm in both groups. Each value represents the mean ± S.E.M. of at
least three independent experiments performed in duplicate.

[35S]GTPγS Saturation Binding Experiments.
[35S]GTPγS saturation binding exhibited high affinities for
G-proteins at MOR (Table 2)
after DAMGO (10 μM) stimulation, but no significant differences were
detectable between Kd Gprotein in DRG membranes of animals
with and without FCA inflammation (p > 0.05)
(Table 2). In animals with FCA
inflammation, a significant increase in DAMGO-stimulated
Bmax Gprotein was detected
(Fig. 4,
Table 2) (p <
0.05). After stimulation with the partial agonist BUP,
Bmax Gprotein was only measurable in animals with FCA
inflammation (Table 2 and
Fig. 4).
Bmax determination of G-proteins in animals with FCA
inflammation revealed that after BUP stimulation, only 34% (145 fmol/mg) of
G-proteins were activated compared with 100% (425 fmol/mg) of G-proteins
activated by the full agonist DAMGO (Table
2, Fig. 4). The
amplification factors (amount of G-protein bound/number of opioid receptors
expressed on the surface) was calculated according to Selley et al.
(1998). No significant
difference in amplification factors was detectable between animals with
(amplification factor 9) and without (amplification factor 11) FCA
inflammation.

Affinity (Kd Gprotein) and number (Bmax
Gprotein) of G-proteins for net agonist-stimulated
[35S]GTPγS binding in DRG membranes of animals without
(Control) and with (FCA) inflammation after DAMGO and BUP stimulation.
Membranes were incubated with varying concentrations of
[35S]GTPγS as described under Materials and Methods.
Data are mean Bmax and Kd values
± S.E.M., obtained from at least three independent experiments.

Saturation analysis of DAMGO and BUP-stimulated [35S]GTPγS
binding to DRG membranes of animals with and without inflammation. Saturation
binding of [35S]GTPγS was performed in the absence and
presence of 10 μM DAMGO or BUP. Unstimulated and stimulated
[35S]GTPγS binding were subtracted.

Behavioral Studies. Intraplantar injection of BUP in a dose of up to
5 μg in normal rats did not show any significant changes in PPT (p
> 0.05) (Fig. 5B). Injection
of higher doses of BUP, such as 10 μg, increased PPT not only in the
injected paw, but also in the contralateral paw of rats with and without FCA
inflammation, indicating a systemic (central) site of action (data not shown).
In contrast, administration of BUP into inflamed paws resulted in significant
elevations of PPT (p < 0.05)
(Fig. 5A). No PPT changes were
observed in the contralateral noninflamed paws (p > 0.05)
indicating that the site of action is restricted to the inflamed paw (data not
shown). PPT elevations in inflamed paws increased dose dependently. BUP in a
dose of 1 μg showed a peak effect at 5 min, whereas BUP in a dose of 3 and
5 μg showed maximum effect at 30 min. This antinociception was very
long-lasting (up to 2 h), and by 4 h, the PPT returned to baseline values
(Fig. 5A). The peripheral
antinociceptive effect produced by BUP in inflamed paws (5 μg at 30 min)
was dose-dependently antagonized by intraplantar coadministration of naloxone
(30 μg, p < 0.05) (Fig.
6A) and CTOP (120 μg, p < 0.05)
(Fig. 6B).

Time course and dose-response of the antinociceptive effects of BUP in
injected paw of (A) rats with inflamed hindpaws and (B) normal rats.
*, significantly different from saline treated group (related to 1,
3, and 5 μg BUP).

Blocking effect of Naloxone (A) and CTOP (B) on the antinociceptive action
of BUP. *, significantly different from saline treated group;!,
significantly different from BUP (5 μg)-treated group.

Discussion

Our results show that inflammation enhances the efficacy of G-protein
coupling of full and partial MOR agonists by an increased binding and
G-protein coupling at MOR of primary afferent neurons. First, FCA inflammation
induces an upregulation of MOR binding sites on DRG membranes without
affecting binding affinities. Consistently, the number of MOR per DRG as well
as the number of MOR-ir neurons that are predominantly of small diameter
increases after FCA inflammation. Second, the full agonist DAMGO but not the
partial agonist BUP induces MOR G-protein coupling in DRG of animals without
FCA inflammation. FCA inflammation results in significant increases in
DAMGO-induced MOR G-protein coupling and in partial agonist activity of
BUP-induced MOR G-protein coupling. Third, BUP injection into inflamed, but
not normal hindpaws elicits potent and long-lasting antinociceptive
effects.

Peripheral opioid receptors are localized and expressed on primary sensory
neurons. Primary sensory neurons offer the advantage of characterizing
receptors in their native environment. In addition, the possibility to induce
a locally applied inflammation allows study of the effects of MOR binding and
signaling under pathological conditions. In animals with FCA inflammation, we
found a significant increase in the number of MOR binding sites on DRG
membranes, although the affinity of DAMGO to MOR remained unchanged.
Displacement experiments of [3H]DAMGO with either nonlabeled DAMGO
or the partial agonist BUP revealed similar inhibitory constants
(Ki) in DRG membranes of animals with and without FCA
inflammation. These results suggest that an inflammatory stimulus can increase
the number of MOR on DRG membranes but does not change the affinity of MOR to
opioids.

Axonal transport has been demonstrated for various neuroreceptors,
including MOR in peripheral nerves (Laduron
and Castel, 1990). We performed a set of experiments to show that
an increase in MOR specific binding sites in the DRG is accompanied by an
increase of the axonal transport of MOR to the periphery. Almost no MOR
binding sites were detectable in the unligated sciatic nerve preparations.
However, ligation of the sciatic nerve in the absence of inflammation resulted
in an accumulation of MOR at the proximal part of the ligation, indicating an
anterograde transport from the DRG to the noninflamed paw. In rats with
inflamed paws, a significant increase in MOR-specific binding sites at the
proximal part of the ligation was detected. Together with our binding data in
DRG membranes, this indicates that inflammation can cause an increase in
MOR-specific binding sites in both DRG and the sciatic nerve. This strongly
suggests that an increase in MOR levels in the DRG would also be seen in the
peripheral portions of the nerve after axonal transport to the periphery. This
also confirms previous neuroanatomical evidence for the existence of MOR in
the sciatic nerve and peripheral cutaneous nerve fibers
(Hassan et al., 1993). It was
shown recently that an increase in MOR number might be related to mediators of
inflammation (e.g., IL-4, tumor necrosis factor)
(Kraus et al., 2001).

Our immunohistochemical results confirmed an up-regulation of MOR after FCA
inflammation. However, this increase was restricted to DRG on the inflamed
side and was not detected in DRG on the contralateral side. An increase in
Bmax (90%, as determined with [3H]DAMGO
binding) and an increase in the number of MOR-positive DRG neurons (45%, as
determined in our immunohistochemical studies) indicates that this
up-regulation is caused by an increase in the number and density of
MOR-positive neurons. Consistent with previous studies, under both normal and
inflammatory conditions, MOR immunoreactive DRG neurons were mainly of small
diameter, suggesting that the increase in MOR immunoreactivity is mainly
restricted to nociceptive neurons (Ji et
al., 1995; Mousa et al.,
2001).

An important finding of the present study is that the efficacy of
DAMGO-stimulated G-protein activation increased significantly in animals with
FCA inflammation. This observation might explain why the application of
exogenous opioids in peripheral antinociception is enhanced under inflammatory
conditions (Stein et al.,
2001). In addition, the mechanisms of μ-opioid agonist efficacy
and inflammation were investigated using agonist-stimulated
[35S]GTPγS binding. The advantages and disadvantages to this
measurements have been described earlier
(Newman-Tancredi et al.,
1997a; Selley et al.,
1997): Scatchard analysis of [35S]GTPγS binding
measures the competition of a radiolabeled ligand
([35S]GTPγS) for a nonlabeled ligand (GDP) only under
nonequilibrium conditions. At equilibrium, the [35S]GTPγS
would displace as much of the GDP as possible, and no agonist-stimulated
binding could be observed. This assay is therefore not quantitatively accurate
in the sense that a given Bmax G protein represents the
exact maximal number of G proteins; however, relative comparisons between
inflamed and noninflamed tissue are possible. We found that the number of
[35S]GTPγS binding sites that can be occupied after DAMGO
stimulation increased 1.6-fold (from 266 to 425 fmol/mg of protein) in animals
with FCA inflammation. The amplification factor (or number of G-proteins
activated per MOR) decreased, suggesting that an increase in MOR during
inflammation is not proportional with an increase in G-protein coupling. In
addition, we found that DAMGO-occupied receptors on DRG membranes in animals
with and without FCA inflammation revealed no differences in the DAMGO-induced
guanine nucleotide affinities (measured as the Kd Gprotein
value of [35S]GTPγS binding). Taken together, these results
suggest that inflammation can cause an increase in receptor density per cell,
which results in an increased number of activated G-proteins. Consistent with
this notion, it was shown earlier that a drop in MOR in SH-SY5Y cells and in
cannabinoid receptors in certain areas in the brain could cause an equivalent
drop in the level of [35S]GTPγS binding
(Sim et al., 1996;
Remmers et al., 2000;
Selley et al., 2001). These
results appear in contrast to other studies in the 5-HT1A system,
where an increase in receptor density did not change the activated G-protein
number (Newman-Tancredi et al.,
1997b). However, those studies were performed in highly expressing
Chinese hamster ovary cells in which the number of G-proteins might be limited
in comparison with the number of receptors. DRG membranes of noninflamed
animals expressed 25 fmol/mg of protein MOR, which is clearly below Chinese
hamster ovary cells expressing 1600 fmol/mg 5-HT1A receptors
(Newman-Tancredi et al.,
1997b). Therefore, an increase of MOR in animals with inflammation
might explain the observed increase in G-protein activation. Although animals
with FCA inflammation exhibited a 90% increase in expression of MOR, maximal
stimulation of [35S]GTPγS binding by DAMGO was only 48%
higher and therefore did not increase proportionally with receptor levels.

Suprisingly, BUP did not induce any detectable G-protein coupling in DRG
membranes of animals without FCA inflammation. In contrast, BUP stimulated
G-protein coupling in DRG membranes of animals with FCA inflammation. The
extent of stimulation of [35S]GTPγS binding (EC50)
in animals with FCA inflammation was lower for BUP than for DAMGO, as in many
other in vitro (Huang et al.,
2001; Zaki et al.,
2000) and in vivo (Gopal et
al., 2002; Traynor et al.,
2002) systems. It has been suggested previously that a given
biological effect requires the switching on (or off) of a certain number of
effector molecules (Chavkin and Goldstein,
1984). Due to stoichiometric interactions between receptors and
effectors, it might be that the partial agonist BUP could not activate a
detectable amount of G-proteins in animals without FCA inflammation. However,
because of the increase of MOR in DRG membranes of animals with FCA
inflammation, the number of receptors appears sufficient to activate the pool
of G-proteins.

Scatchard analysis of basal and agonist-stimulated
[35S]GTPγS binding confirmed that BUP (low efficacy partial
agonist) produced a lower affinity GTP-binding state in DRG of animals with
inflammation (presumably in the guanine nucleotide binding site of μ
receptor-coupled G protein α subunits) than DAMGO (higher efficacy
agonist). Partial agonists do not fully shift the affinity of the G-proteins
into a GTP-preferring state (Selley et
al., 1998; Traynor et al.,
2002), and this was clearly evident for BUP in DRG membranes of
animals with FCA inflammation: Agonist-induced guanine nucleotide affinity for
BUP (Kd Gprotein, 1.8 nM) compared to DAMGO
(Kd Gprotein, 0.8 nM) was different and the catalytic
activation of G proteins, as measured by the agonist-stimulated
Bmax Gprotein of [35S]GTPγS binding, was
lower for BUP (Bmax Gprotein, 145fmol/mg) than for DAMGO
(Bmax Gprotein, 425fmol/mg).

It should be noted that an increase in maximal [35S]GTPγS
binding as a function of receptor density does not necessarily result in a
similar increase in the magnitude of a downstream response
(Law et al., 1994;
Prather et al., 1994).
Therefore, we performed a set of behavioral experiments to test whether the
results obtained with [35S]GTPγS binding and BUP showed
functional consequences in antinociception.

We found that intraplantar injection of the partial agonist BUP in
noninflamed paws did not change paw pressure thresholds compared with
intraplantar saline injections. In contrast, local BUP injections in animals
with inflamed paws produced PPT elevations (i.e., antinociception). These
results indicate that BUP can act as an effective peripheral antinociceptive
agent only in the presence of inflammation. The MOR-selective antagonists NLX
and CTOP could block this antinociceptive effect of BUP, which clearly
indicates that BUP mediates its antinociceptive activity through MOR. The
contralateral paws showed no changes in PPT, suggesting that low doses of BUP
induce only peripheral, not central, opioid analgesic effects. It was already
shown earlier that opioid full agonists (e.g., fentanyl) produce
dose-dependent elevations of PPT in animals with and without FCA inflammation;
however, antinociception is smaller in noninflamed hindpaws compared with
inflamed hindpaws (Antonijevic et al.,
1995). There are many steps between MOR binding and
antinociception (e.g., inhibition of cAMP, inhibition of calcium channel
conductance) that can modulate the downstream responses. However, the observed
antinociceptive action after BUP injection only in animals with FCA
inflammation might be related to the lack of G-protein coupling observed in
DRG membranes. This supports the hypothesis that in animals without FCA
inflammation, there are not enough receptors present to develop opioid
analgesia by the partial MOR agonist BUP.

In conclusion, inflammation is associated with an up-regulation of MOR,
mainly in small-sized primary afferent neurons, and enhances the efficacy of
full and partial MOR agonists in G-protein coupling. These changes might
contribute to the occurrence of peripheral antinociceptive effects of the
partial MOR agonist BUP, which are not present under normal conditions. These
adaptive changes underscore the important differences in opioid receptor
binding and signaling between normal and inflamed tissue. They strongly
indicate that clinical studies testing peripherally active opioids are much
more likely to yield positive results when they are performed in inflammatory
painful conditions.

Footnotes

This work was supported by “Klinische Forschergruppe Grant from the
Deutsche Forschungsgemeinschaft KFO 100/1”.

Gopal S, Tzeng TB, and Cowan A (2002) Characterization
of the pharmacokinetics of buprenorphine and norbuprenorphine in rats after
intravenous bolus administration of buprenorphine. Eur J Pharm
Sci15:287–293.

Hassan AH, Ableitner A, Stein C, and Herz A (1993)
Inflammation of the rat paw enhances axonal transport of opioid receptors in
the sciatic nerve and increases their density in the inflamed tissue.
Neuroscience55:185–195.

Stein C, Millan MJ, and Herz A (1988b) Unilateral
inflammation of the hindpaw in rats as a model of prolonged noxious
stimulation: alterations in behavior and nociceptive thresholds.
Pharmacol Biochem Behav31:455–461.